CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti

To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repai...

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Autores principales: Chae, Keun, Overcash, Justin M., Dawson, Chanell, Valentin, Collin, Tsujimoto, Hitoshi, Myles, Kevin M., Adelman, Zach N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10357993/
https://www.ncbi.nlm.nih.gov/pubmed/37475832
http://dx.doi.org/10.1016/j.crbiot.2023.100133
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author Chae, Keun
Overcash, Justin M.
Dawson, Chanell
Valentin, Collin
Tsujimoto, Hitoshi
Myles, Kevin M.
Adelman, Zach N.
author_facet Chae, Keun
Overcash, Justin M.
Dawson, Chanell
Valentin, Collin
Tsujimoto, Hitoshi
Myles, Kevin M.
Adelman, Zach N.
author_sort Chae, Keun
collection PubMed
description To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), is also critical for efficient gene editing and for homing-based gene drive approaches developed for the control of disease-transmitting vector mosquitoes. However, little is understood about DNA repair homeostasis in the mosquito genome. Here, we utilized CRISPR/Cas9 to generate indel mutant strains for core NHEJ factors ku80, DNA ligase IV (lig4), and DNA-PKcs in the mosquito Aedes aegypti and evaluated the corresponding effects on DNA repair. In a plasmid-based assay, disruption of ku80 or lig4, but not DNA-PKcs, reduced both NHEJ and SSA. However, a transgenic reporter strain-based test revealed that those mutations significantly biased DNA repair events toward SSA. Interestingly, ku80 mutation also significantly increased the end joining rate by a yet-characterized mechanism in males. Our study provides evidence that the core NHEJ factors have an antagonistic effect on SSA-based DSB repair of the Ae. aegypti genome. Down-modulating the NHEJ pathway can enhance the efficiency of nuclease-based genetic control approaches, as most of those operate by homology-based repair processes along with extensive DNA end resection that is antagonized by NHEJ.
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spelling pubmed-103579932023-07-20 CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti Chae, Keun Overcash, Justin M. Dawson, Chanell Valentin, Collin Tsujimoto, Hitoshi Myles, Kevin M. Adelman, Zach N. Curr Res Biotechnol Article To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), is also critical for efficient gene editing and for homing-based gene drive approaches developed for the control of disease-transmitting vector mosquitoes. However, little is understood about DNA repair homeostasis in the mosquito genome. Here, we utilized CRISPR/Cas9 to generate indel mutant strains for core NHEJ factors ku80, DNA ligase IV (lig4), and DNA-PKcs in the mosquito Aedes aegypti and evaluated the corresponding effects on DNA repair. In a plasmid-based assay, disruption of ku80 or lig4, but not DNA-PKcs, reduced both NHEJ and SSA. However, a transgenic reporter strain-based test revealed that those mutations significantly biased DNA repair events toward SSA. Interestingly, ku80 mutation also significantly increased the end joining rate by a yet-characterized mechanism in males. Our study provides evidence that the core NHEJ factors have an antagonistic effect on SSA-based DSB repair of the Ae. aegypti genome. Down-modulating the NHEJ pathway can enhance the efficiency of nuclease-based genetic control approaches, as most of those operate by homology-based repair processes along with extensive DNA end resection that is antagonized by NHEJ. 2023 2023-05-29 /pmc/articles/PMC10357993/ /pubmed/37475832 http://dx.doi.org/10.1016/j.crbiot.2023.100133 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) ).
spellingShingle Article
Chae, Keun
Overcash, Justin M.
Dawson, Chanell
Valentin, Collin
Tsujimoto, Hitoshi
Myles, Kevin M.
Adelman, Zach N.
CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti
title CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti
title_full CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti
title_fullStr CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti
title_full_unstemmed CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti
title_short CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti
title_sort crispr-based gene editing of non-homologous end joining factors biases dna repair pathway choice toward single-strand annealing in aedes aegypti
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10357993/
https://www.ncbi.nlm.nih.gov/pubmed/37475832
http://dx.doi.org/10.1016/j.crbiot.2023.100133
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